A common task in molecular biology is to identify and quantify the presence of a protein in a complex mixture. For example, to identify the level of expression of a protein of interest, a western blot can be performed in which a protein extract is run on a gel and stained with antibodies against a defined epitope of the protein of interest. The defined epitope can be a particular sequence or structure found in the native protein, or can be a tag introduced during cloning, e.g., a “FLAG tag” for which specific antibodies are commercially available. A secondary peroxidase-conjugated antibody specific for the primary antibody bound to the protein is used to generate a detectable signal. This process is cumbersome and more streamlined methods for purification of proteins from cell extracts are desirable.
Attaching optical labels to proteins can be an alternate strategy for detection and quantification, however, this typically requires chemical modification of residues within the protein. Attaching dyes through lysine or cysteine residues often modifies activity or reduces solubility making purification of the labeled protein difficult or impossible. Fluorescent protein tags are available with a wide variety of spectral properties, e.g., as described in Shaner, et al. (2005) Nature Methods 2(12):905-909, incorporated by reference herein in its entirety for all purposes, but these tags are suboptimal for single-molecule experimentation.
The ability to synthesize DNA chemically has made possible the construction of peptides and proteins not otherwise found in nature and useful in a wide variety of methods that would otherwise be very difficult or impossible to perform. The patent literature, for instance, is replete with publications describing the recombinant expression of receptor proteins. See, e.g., PCT Patent Pub. No. 91/18982 and U.S. Pat. Nos. 5,081,228 and 4,968,607, which describe recombinant DNA molecules encoding the IL-1 receptor; U.S. Pat. Nos. 4,816,565; 4,578,335; and 4,845,198, which describe recombinant DNA and proteins relating to the IL-2 receptor; PCT Patent Pub. No. 91/08214, which describes EGF receptor gene related nucleic acids; PCT Patent Pub. No. 91/16431 and U.S. Pat. No. 4,897,264, which describe the interferon gamma receptor and related proteins and nucleic acids; European Patent Office (EPO) Pub. No. 377,489, which describes the C5a receptor protein; PCT Patent Pub. No. 90/08822, which describes the EPO receptor and related nucleic acids; PCT Patent Pub. No. 92/01715, which describes MHC receptors; and U.S. patent application Ser. No. 947,339, filed on Sep. 18, 1992, which describes how HPAP-containing receptors can be cleaved from the cell surface and how the anchoring sequences that remain can serve as recognition sequences for antibodies that are used to immobilize the receptor. Several of these publications, each of which is incorporated herein by reference for all purposes, describe both how to isolate a particular receptor protein (or the gene encoding the protein) and variants of the receptor that may be useful in ways the natural or native receptor is not.
The advances made with respect to receptor cloning and expression have been accompanied by advances in technology relating to methods for screening a receptor against compounds that may interact with the receptor in a desired fashion. One such advance relates to the generation of large numbers of compounds, or potential ligands, in a variety of random and semi-random “peptide diversity” generation systems. These systems include the “peptides on plasmids” system described in U.S. Pat. No. 5,338,665, which is a continuation-in-part of U.S. Pat. No. 5,270,170; the “peptides on phage” system described in U.S. patent application Ser. No. 718,577, filed Jun. 20, 1991, which is a continuation-in-part of Ser. No. 541,108, filed Jun. 20, 1990; Cwirla et al., August 1990, Proc. Natl. Acad. Sci. USA 87: 6378-6382; Barrett et al., 1992, Analyt. Biochem. 204: 357-364; and PCT Patent Pub. Nos. 91/18980 and 91/19818; the phage-based antibody display systems described in U.S. patent application Ser. No. 517,659, filed May 11, 1990, and PCT Patent Pub. No. 91/17271; the bead-based systems for generating and screening nucleic acid ligands described in PCT Pub. Nos. 91/19813, 92/05258, and 92/14843; the bead-based system described in U.S. patent application Ser. No. 946,239, filed Sep. 16, 1992, which is a continuation-in-part of Ser. No. 762,522, filed Sep. 18, 1991; and the “very large scaled immobilized polymer synthesis” system described in U.S. Pat. No. 5,143,854; PCT Patent Pub. Nos. 90/15070 and 92/10092, U.S. patent application Ser. No. 624,120, filed Dec. 6, 1990; Fodor et al., Feb. 15, 1991, Science 251: 767-773; Dower and Fodor, 1991, Ann. Rep. Med. Chem. 26:271-180; and U.S. patent application Ser. No. 805,727, filed Dec. 6, 1991. Each of the above references is incorporated herein by reference for all purposes.
Other developments relate to how the receptor is used in such screening methods. One important advance relates to the development of reagents and methods for immobilizing one or more receptors in a spatially defined array, as described in PCT Patent Pub. No. 91/07087, which describes attachment of a receptor to avidin and subsequent immobilization on a surface that bears biotin groups. Once the avidinylated receptor is bound to the biotin groups on the surface, the surface can be used in screening compounds against the receptor.
Biotin is a cofactor that is covalently attached to several enzymes involved in the transfer of activated carboxyl groups. Biotin labeling of molecules not normally biotinylated can be used to label, detect, purify, and/or immobilize such molecules. These methods also rely upon the proteins avidin and/or streptavidin, which bind very tightly and specifically to biotin. Typically, the biotinylated molecules used in such methods are prepared by an in vitro biotinylation process. Alternatively, methods for biotinylating proteins synthesized by recombinant DNA techniques in vivo eliminates the need to chemically biotinylate these proteins after purification and greatly simplifies the purification process, due to the ability to use the biotin as an affinity tag (see Green, 1975, Adv. Protein Res. 29:85-133, incorporated herein by reference).
Biotin-streptavidin interactions can also be used for linking different molecules together to form useful complexes. For example, since streptavidin has four binding sites for biotin, four biotin-labeled molecules can be linked to a single streptavidin molecule. Certain specific examples of methods comprising linkage of biotin-labeled molecules through a streptavidin molecule are described in detail in the art, e.g., in U.S. patent application Ser. No. 13/767,619, filed Feb. 14, 2013; U.S. Pat. Nos. 8,389,676; and 8,252,910, all of which are incorporated herein by reference in their entireties for all purposes. However, for some applications it is useful to generate a strong 1:1 complex of two molecules, and this can be difficult with streptavidin due to its tetravalent nature. Many different stoichiometries can be generated, e.g., 1:3 and 3:1. Methods have been previously described for creating streptavidin tetramers with reduced numbers of active sites, e.g., in Howarth, et al. (2006) Nature Methods 3(4):267-73, which is incorporated herein by reference in its entirety for all purposes. However, the methods of Howarth involve mixing of two species of recombinant streptavidin and are cumbersome.